CN113209285A

CN113209285A - Application of helicobacter pylori chemotactic factor chemotaxis gene

Info

Publication number: CN113209285A
Application number: CN202110436351.7A
Authority: CN
Inventors: 杜方川; 童文德; 朱白梅; 童武学; 杨融融
Original assignee: Chengdu Yimiao Biotechnology Co ltd
Current assignee: Chengdu Olymvax Biopharmaceuticals Inc
Priority date: 2021-04-22
Filing date: 2021-04-22
Publication date: 2021-08-06
Anticipated expiration: 2041-04-22
Also published as: CN113209285B

Abstract

The invention discloses an application of a helicobacter pylori chemotactic factor chemotaxis gene in preparation of a helicobacter pylori vaccine, wherein the nucleotide sequence of the chemotaxis gene is shown as SEQ ID NO. 1.

Description

Application of helicobacter pylori chemotactic factor chemotaxis gene

Technical Field

The invention relates to the technical field of genetic engineering, in particular to application of a helicobacter pylori chemotactic factor chemotaxis gene.

Background

Helicobacter pylori (Hp) is a gram-negative bacterium, which often causes digestive system diseases such as chronic gastritis, peptic ulcer, gastric lymphoproliferative lymphoma, gastric cancer and the like of patients after infecting people. At present, no helicobacter pylori vaccines in the market are available at home and abroad, but 6 helicobacter pylori vaccines including Novartis are sequentially subjected to clinical experiments, including inactivated whole-bacterium vaccines, recombinant subunit vaccines, recombinant multi-subunit vaccines and vector vaccines.

Up to now, there have been published sequencing strains of helicobacter pylori having more than 200 strains, more than 1400 proteins, and 20% -30% genome difference among different strains, and 273 candidate antigens of helicobacter pylori were preliminarily selected by relevant biological software, wherein chemokines were of great interest for development as candidate vaccines of helicobacter pylori.

Chemokines are a class of small cytokines or signaling proteins that have the ability to induce directional chemotaxis of nearby responding cells. For example, when a human body defends and removes foreign matters such as invading pathogens, some substances can lead immune cells to perform directional chemotaxis, such as SIS cytokines and the like. During the colonization process of helicobacter pylori, the chemotactic signal system regulates the movement of helicobacter pylori, so that the helicobacter pylori passes through the gastric mucus layer from gastric juice and then reaches the basal layer from the epithelial layer of the gastric mucosa. Studies have shown that the rate of helicobacter pylori colonization is reduced in chemotactic system-deficient mutants. The application provides a theoretical basis for research of the chemotactic factor as a candidate antigen of the helicobacter pylori vaccine and provides a new breakthrough for the development of the helicobacter pylori vaccine.

Therefore, how to apply the helicobacter pylori chemotactic factor to the preparation of the helicobacter pylori vaccine is a problem which needs to be solved urgently by the technical personnel in the field.

Disclosure of Invention

In view of the above, the invention provides an application of a helicobacter pylori chemotactic factor chemotaxis protein, wherein the chemotaxis protein can be applied to preparation of a helicobacter pylori vaccine, the protection rate is up to 20.7%, and a guarantee is provided for preventing helicobacter pylori infection.

In order to achieve the purpose, the invention adopts the following technical scheme:

an application of a helicobacter pylori chemotactic factor chemotaxis gene in preparing a helicobacter pylori vaccine, wherein the nucleotide sequence of the chemotaxis gene is shown as SEQ ID NO. 1.

An application of helicobacter pylori chemotactic factor chemotaxis recombinant protein in preparing helicobacter pylori vaccine, wherein the amino acid sequence of the chemotaxis recombinant protein is shown as SEQ ID NO. 2.

The technical effect achieved by the technical scheme is as follows: the chemotactic factor chemotaxis protein is applied to the preparation of the helicobacter pylori vaccine for the first time, and the popularization and application of the helicobacter pylori are guaranteed.

The Chemotaxis recombinant protein has good immunogenicity, can induce a mouse to generate mucosal immune response, can eliminate the colonization of helicobacter pylori SS1 strains in the stomach of the mouse, and can be prepared into a helicobacter pylori subunit vaccine by a mucosal adjuvant and the Chemotaxis recombinant protein to prevent helicobacter pylori infection.

An expression vector for expressing helicobacter pylori chemotactic factor chemotaxis recombinant protein, which comprises: chemotaxis recombinant protein, His-tag, pET29b plasmid, GST protein and MBP protein.

A construction method of an expression vector for expressing helicobacter pylori chemotactic factor chemotaxis recombinant protein comprises the following steps:

1) connecting the His-tag gene, the MBP gene and the GST gene to obtain the His-tag-MBP-GST gene;

2) connecting the His-tag-MBP-GST gene obtained in the step 1) with pET29b plasmid, and transforming competent cells to obtain pET29b-5 plasmid;

3) cloning a chemitaxis gene, connecting the cloned chemitaxis gene with pET29b-5 plasmid obtained in the step 2), and transforming competent cells to obtain an expression vector of the chemitaxis recombinant protein.

A fermentation method for expressing helicobacter pylori chemotactic factor chemotaxis recombinant protein comprises the following steps:

1) pET29b-5-Chemotaxis Escherichia coli inoculation culture: taking the constructed pET29b-5-Chemotaxis escherichia coli engineering bacteria out of an ultralow temperature refrigerator at minus 80 ℃, inoculating the engineering bacteria into an LB-1 culture medium, and culturing overnight at constant temperature of 37 ℃ and 220 rpm;

2) e.coli expansion culture of pET29 b-5-Chemotaxis: taking out the engineering bacteria cultured overnight, inoculating the engineering bacteria to a TB culture medium according to the inoculation ratio of 1.5%, inoculating 4L of engineering bacteria, and culturing at constant temperature of 37 ℃ and 220rpm for 3 h;

3) pET29b-5-Chemotaxis escherichia coli induced expression: when the amplification culture is finished, setting the induction temperature to be 15 ℃, and adding 0.08mM IPTG for induction expression, wherein the induction time is 20 h;

4) pET29b-5-Chemotaxis Escherichia coli thallus collection: and (3) when induction is finished, centrifugally collecting thalli by using a centrifugal machine, and then breaking the thalli to obtain the recombinant protein.

As a preferred technical scheme of the invention, the TB culture medium comprises the following components: 0.2312% of monopotassium phosphate, 1.2540% of dipotassium phosphate, 2.4000% of yeast extract, 1.2000% of tryptone, 0.4000% of glycerol, 0.0500% of an antifoaming agent and the balance of water.

As a preferred technical scheme of the invention, the centrifugation parameters in the step 4) are as follows: 10000g, 8 ℃, 10 min.

According to the technical scheme, compared with the prior art, the recombinant protein of the helicobacter pylori Chemotaxis is cloned and expressed by adopting a genetic engineering technology, the expression of the supernatant is high, the separation and purification steps are simple and convenient, the immunity valence is high, and the protection is realized. The Chemotaxis recombinant protein can be directly mixed with mucosal adjuvant LT (B)₅The composition can be used in combination for oral immunization.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a schematic diagram showing the construction of pET29b-5 plasmid;

FIG. 2 is a diagram showing the results of plasmid identification PCR for His (tag) -MBP-GST gene fragment; m is DNA molecular weight standard; 1 is His (tag) -MBP-GST gene fragment (1803bp) PCR product;

FIG. 3 is the result of high fidelity PCR amplification of Chemotaxis gene fragment; m is DNA molecular weight standard; 1 is a PCR product of a Chemotaxis gene fragment (936 bp);

FIG. 4 is a diagram showing the result of general PCR identification of recombinant pET29b-Chemotaxis/BL21(DE)3, wherein M is a DNA molecular weight standard; 1 is a PCR product of a Chemotaxis gene fragment (936 bp);

FIG. 5 is a schematic diagram of affinity chromatography of Chemotaxis recombinant protein/BL 21(DE)3 engineering bacteria broken supernatant after primary purification, wherein the first peak is a broken supernatant sample peak, the second, third and fourth peaks are A2 buffer solution washing impurity protein peaks, the fifth peak is an A3 buffer solution washing impurity protein peak, and the sixth peak is an A4 buffer solution elution target protein peak;

FIG. 6 is a drawing showing the results of induction expression and supernatant purification of recombinant engineering bacteria of Chemotaxis recombinant protein/BL 21(DE)3, wherein lane 1 is protein molecular mass standard (Thermo Fisher, 26616), lane 2 is the whole bacteria of Chemotaxis/BL21(DE)3 recombinant engineering bacteria, lane 3 is the supernatant of Chemotaxis/BL21(DE)3 recombinant engineering bacteria, and lane 4 is the first purification flow-through of the Chemotaxis/BL21(DE)3 recombinant engineering bacteria; lane 5 is a second flow-through of the supernatant of recombinant engineering bacteria Chemotaxis/BL21(DE) 3; lane 6 is a Chemotaxis recombinant protein containing a solubility-promoting tag; lane 7 is a Chemotaxis recombinant protein containing a solubility-promoting tag digested with PP enzyme; lane 8 is Chemotaxis recombinant protein;

FIG. 7 shows recombinant pET28a-LT (B)₅/BL21(DE)₃Common PCR identification results; m is DNA molecular weight standard; 1 is LT (B)₅Gene fragment (375bp) PCR product;

FIG. 8 is the drawing LT (B)₅/BL21(DE)₃Performing primary purification on the engineering bacteria-broken supernatant by using an affinity chromatography; the first peak is a broken bacteria supernatant sample peak, and the second peak is B₂Eluting with buffer solution to obtain target protein peak;

FIG. 9 is LT (B)₅/BL21(DE)₃Inducing expression of the recombinant engineering bacteria, and purifying the supernatant after bacteria breaking; lane 1 is LT (B)₅/BL21(DE)₃The engineering bacteria are broken into whole bacteria, lane 2 is LT (B)₅/BL21(DE)₃The supernatant of the engineering bacteria is LT (B) in lane 3₅/BL21(DE)₃The engineering bacteria is broken and deposited, lane 4 is LT (B)₅/BL21(DE)₃The supernatant of the engineered bacteria is purified and first flowed through, and a lane 5 is LT (B)₅/BL21(DE)₃The supernatant of the engineered bacteria was purified and passed through for the second time, lane 6 was protein molecular mass standard (Thermo Fisher, 26616), lane 7 was LT (B)₅A recombinant protein;

FIG. 10 shows the positive conversion rate of mouse saliva IgA (1:4) and serum IgG (1:800) after a Balb/c mouse is immunized with Chemotaxis recombinant protein.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: construction of pET29b-5 double-dissolving-promoting-label vector

This example provides a genetic construction, fermentation and purification method for the Chemotaxis protein, a chemotactic factor for helicobacter pylori.

The invention provides a recombinant expression vector for expressing a Chemotaxis recombinant protein, which comprises a nucleotide sequence and a plasmid sequence for encoding the Chemotaxis recombinant protein, His (tag), a lysogenic protein, wherein the plasmid is preferably pET29b, the His (tag) sequence is 7 histidines (His), and the lysogenic protein sequence is divided into GST and MBP (maltose binding protein). The plasmid was constructed as shown in FIG. 1.

His (tag), MBP and GST gene clone and connection

1. The amino acid sequence of the protein of His (tag) is selected from SEQ ID NO.3, and the nucleotide sequence is shown as SEQ ID NO. 4.

2. The MBP gene is derived from pMAL-c2X plasmid (Changsha Youbao biotechnology, Co., Ltd.), the protein amino acid sequence of the MBP is selected from SEQ ID NO.5, and the nucleotide sequence is shown as SEQ ID NO. 6.

3. The GST gene is derived from pGEX-6P-1 plasmid (Changsha Youbao Biotechnology Co., Ltd.), the protein amino acid sequence of GST is selected from SEQ ID NO.7, and the nucleotide sequence is shown as SEQ ID NO. 8.

4. Designing a corresponding primer according to the primer design principle, and adding an enzyme cutting site. The primer sequences are shown in table 1:

TABLE 1

5. His (tag), MBP and GST gene connection

His (tag), MBP (MBP) and GST gene sequences are amplified by a high fidelity PCR method and the primer sequences in the table 1, and a high fidelity system and a program refer to an instruction book. High fidelity PCR enzyme (KOD-Plus-Neo) was from Shanghai Biotech, Toyobo.

The high fidelity PCR was performed using pMAL-c2X plasmid as the template and F29b-1, R502 as the primers, and the product was named MBP-1.

pGEX-6P-1 plasmid is used as a template, F502 and R503 are used as primers to carry out high fidelity PCR, and the product is named as GST-1.

MBP-1 and GST-1 are taken as templates, F29b-2 and R503 are taken as primers to carry out high fidelity PCR, and the product is named as His (tag) -MBP-GST, and the gene size of the His (tag) -MBP-GST is 1803 bp.

Carrying out 1.5% agarose gel electrophoresis on the product after high-fidelity PCR, wherein the electrophoresis parameters are 220V and 30min, and observing the result under an imaging system after the electrophoresis is finished;

and when the high-fidelity PCR fragment is consistent with the size of the original sequence fragment, carrying out agarose gel recovery, wherein the gel recovery operation refers to the instruction. The gel recovery was performed using agarose gel recovery kit (Shanghai Czeri bioengineering, Inc.).

(II) His (tag) -MBP-GST gene is connected with pET29b plasmid

His (tag) -MBP-GST gene and pET29b were each digested at the sites of NedI/NcoI. The enzyme digestion system and the procedure are referred to the description. NedI/NcoI was from Baori physicians technology (Beijing) Inc.

And (3) performing 1% agarose gel electrophoresis on the products subjected to double enzyme digestion respectively, and observing the result under an imaging system after the electrophoresis is finished. The desired fragments were recovered by cutting, and then agarose gel recovery was performed as described above. The double-digested His (tag) -MBP-GST gene and pET29b plasmid were ligated by using ligase, and the ligation system and the procedure were as described in the specification. The ligated product was named pET29b-5, and ligase (Ligation high) was obtained from Shanghai Biotech Co., Ltd.

(III) pET29b-5 plasmid transformation DH5a

The ligated product pET29b-5 plasmid was transformed into E.coli DH5a competent cells, and the transformation system and procedures were as described in the specification. Coli DH5a competent cells were obtained from Baori physician technology (Beijing) Ltd.

The transformation product was added to 500. mu.L of LB liquid and cultured with shaking at 220rpm at 37 ℃ for 1 hour, which was culture 1. The LB medium consisted of: yeast extract 0.500%, tryptone 1.000%, sodium chloride 1.000%, solvent water.

100 μ L of culture 1 was spread on LB agar plates and cultured overnight at 37 ℃. The LB agar plate consists of: yeast extract 0.500%, tryptone 1.000%, sodium chloride 1.000%, agar powder 1.750%, kanamycin 0.001%, and water as solvent.

Colonies were picked on overnight-cultured LB agar plates and subjected to general PCR identification, and the identification system and procedure were as described in TaKaRa Ex Taq (TaKaRa Ex Taq) kit, primers F29b-2 and R503, TaKaRa Ex Taq was from Baori physician technology (Beijing) Ltd.

Mixing 10 mu L of colony PCR amplification solution with 10 Xloadingbuffer 1 mu L, performing 1.5% agarose gel electrophoresis with electrophoresis parameters of 220V and 30min, and observing the result under an imaging system after the electrophoresis is finished.

The colony PCR-identified positive bacteria were inoculated in LB-1 medium and cultured overnight at 37 ℃ with shaking at 220rpm, this being culture 2. The LB-1 medium consists of: yeast extract 0.500%, tryptone 1.000%, sodium chloride 1.000%, kanamycin 0.001%, and solvent water.

And (3) performing seed preservation on the culture 2, wherein the seed preservation parameters are that the culture 2: the ratio of 30% glycerol was 1:1, and the storage conditions were-80 ℃.

(IV) identification of pET29b-5 plasmid (containing His (tag) -MBP-GST gene)

The culture 2 was subjected to plasmid extraction, and the plasmid extraction procedure was as described in the specification. The recombinant plasmid DNA extraction kit is from Shanghai Czeri bioengineering Co., Ltd.

The extracted plasmid is subjected to common PCR identification, the primers are F29b-2 and R503, and the identification system and the program refer to TaKaRa Ex Taq instructions.

Mixing 10. mu.L of plasmid PCR amplification solution with 10 × loadingbuffer 1. mu.L, performing 1.5% agarose gel electrophoresis with electrophoresis parameters of 220V for 30min, and observing the result under an imaging system after the electrophoresis is finished, wherein the result is shown in FIG. 2.

The plasmid which is identified by common PCR is sequenced, sequencing primers are a T7 promoter and a T7 terminator, and the sequencing unit is the company of the engineering bioengineering (Shanghai) GmbH.

And comparing the sequencing result with the original sequence, wherein the coincidence rate is 100%.

The sequencing-compatible plasmid was stored at-20 ℃ for future use.

Example 2: chemotaxis gene construction

Cloning of Chemotaxis Gene

The strain of helicobacter pylori SS1 is from the Chinese food and drug testing institute.

The culture of helicobacter pylori refers to the conventional conditions, formula and operation method in the field. Taking helicobacter pylori culture to extract whole genome, referring to the extraction system and program, and storing the extracted product at-20 ℃ for later use. The bacterial genome extraction kit is from Shanghai Czeri bioengineering GmbH.

According to the principle of primer design, corresponding primers of the Chemotaxis gene are designed, and enzyme cutting sites NcoI and EcoRI are added. The primer sequences are shown in table 2:

TABLE 2

The high-fidelity PCR method is adopted to amplify the gene sequence, the Chemotaxis gene size is 936bp, and the high-fidelity PCR enzyme, the high-fidelity system and the program are the same as above.

And (3) carrying out 1.5% agarose gel electrophoresis on the high-fidelity product, wherein the electrophoresis parameters are 220V and 35min, and observing the result under an imaging system after the electrophoresis is finished, wherein the result is shown in figure 3.

And when the high-fidelity PCR fragment is consistent with the original sequence fragment in size, carrying out agarose gel recovery, wherein the gel recovery operation is the same as the above.

(II) the Chemotaxis gene is connected with pET29b-5 plasmid

The Chemotaxis gene and pET29b-5 are subjected to double enzyme digestion respectively, and the enzyme digestion sites are NcoI and EcoRI. Enzyme digestion System and procedure with reference to the description NcoI/EcoRI enzyme was from Baozi physician's technology (Beijing) Ltd.

And (3) performing 1% agarose gel electrophoresis on the products subjected to double enzyme digestion respectively, and observing the result under an imaging system after the electrophoresis is finished.

The desired fragment was recovered by cutting the gel and agarose gel recovery was performed as described above.

The double-enzyme-cleaved Chemotaxis gene and the pET29b-5 plasmid are connected by using ligase, and the source of the ligase, the enzyme digestion system and the procedure are the same as above.

(III) pET29b-5-Chemotaxis transforming DH5a

The ligated product was transformed into E.coli DH5a competent cells, and the transformation system and procedure were as described in the specification, E.coli DH5a competent cells were obtained from Baori physician technology (Beijing) Co., Ltd.

The transformation product was added to 500. mu.L of LB liquid and cultured with shaking at 220rpm at 37 ℃ for 1 hour, which was culture 3. The composition of LB medium was the same as above.

100 μ L of culture 3 was spread on LB agar plates and cultured overnight at 37 ℃. LB agar plates were composed as above.

Colonies were picked on overnight-cultured LB agar plates and subjected to general PCR identification using primers F93-a and R93, and the identification system and procedure were as described in TaKaRa Ex Taq (TaKaRa Ex Taq from Baozi Biotech technology (Beijing) Ltd.).

The colony PCR-identified positive bacteria were inoculated in LB-1 medium and cultured overnight at 37 ℃ with shaking at 220rpm, this being culture 4. The composition of LB-1 medium is the same as above.

And (3) performing seed preservation on the culture 4, wherein the seed preservation parameters are that the culture 4: the ratio of 30% glycerol was 1:1, and the storage conditions were-80 ℃.

(IV) extraction of pET29b-5-Chemotaxis plasmid

The overnight culture 4 was extracted with the plasmid designated pET29 b-5-Chemotaxis. The heavy plasmid extraction procedure was as described in the specification. The recombinant plasmid DNA extraction kit is from Shanghai Czeri bioengineering Co., Ltd.

The extracted plasmid is identified by common PCR, the primers are F93-a and R93, and the identification system and the program refer to TaKaRa Ex Taq instructions.

Mixing 10 μ L of plasmid identification common PCR amplification solution with 10 × loading buffer 1 μ L, performing 1.5% agarose gel electrophoresis with electrophoresis parameters of 220V for 30min, and observing the result under an imaging system after the electrophoresis is finished.

The plasmid (800-1000bp) identified by ordinary PCR is stored at-20 ℃ for later use.

(V) pET29b-5-Chemotaxis transformed BL21(DE)₃

pET29b-5-Chemotaxis plasmid was transferred into E.coli BL21(DE)₃Competent cell, E.coli BL21(DE)₃Competent cells were obtained from Baori physician technology (Beijing) Ltd, and the transformation system and procedures were described in the specification.

The transformation product was added to 500. mu.L of LB liquid and cultured with shaking at 220rpm at 37 ℃ for 1 hour, which was culture 5. The composition of LB medium was the same as above.

50 μ L of culture 5 was spread on LB agar plates and incubated overnight at 37 ℃. LB agar plates were composed as above.

Colonies were picked on overnight-cultured LB agar plates for general PCR identification, and TaKaRa Ex Taq source, general PCR identification system and program were referred to TaKaRa Ex Taq instructions.

Mixing 10 μ L of colony PCR amplification solution with 10 × loadingbuffer 1 μ L, performing 1.5% agarose gel electrophoresis with electrophoresis parameters of 220V for 30min, and observing the result under an imaging system after the electrophoresis is finished, wherein the result is shown in FIG. 4.

The colony PCR-identified positive bacteria were inoculated in LB-1 medium and cultured overnight at 37 ℃ with shaking at 220rpm, this being culture 6. The composition of LB-1 medium is the same as above.

Culture 6 was seed protected with overnight culture: the volume ratio of 30% glycerol is 1:1, and the storage condition is-80 ℃.

Example 3: chemotaxis Escherichia coli fermentation

(I) pET29b-5-Chemotaxis escherichia coli inoculation culture

Taking the constructed pET29b-5-Chemotaxis escherichia coli engineering bacteria (culture 6) out of an ultra-low temperature refrigerator at the temperature of-80 ℃, inoculating the engineering bacteria into an LB-1 culture medium, and culturing at the constant temperature of 37 ℃ and 220rpm overnight. The composition of LB-1 medium is the same as above.

(II) pET29b-5-Chemotaxis escherichia coli amplification culture

The engineering bacteria cultured overnight are taken out, inoculated in a TB culture medium according to the inoculation ratio of 1.5 percent, inoculated with 4L together, and cultured at constant temperature of 37 ℃ and 220rpm for 3 h. The TB medium consists of: 0.2312% of monopotassium phosphate, 1.2540% of dipotassium phosphate, 2.4000% of yeast extract, 1.2000% of tryptone, 0.4000% of glycerol, 0.0500% of antifoaming agent and water as solvent.

(III) pET29b-5-Chemotaxis escherichia coli induced expression

When the amplification culture is finished, the induction temperature is set to be 15 ℃, 0.08mM IPTG is added for induction expression, and the induction time is 20 h.

(IV) pET29b-5-Chemotaxis Escherichia coli thallus collection

And (3) when induction is finished, centrifugally collecting thalli by using a centrifugal machine, wherein centrifugal parameters are as follows: 10000g, 8 ℃, 10min, and after the centrifugation, the thalli are stored at minus 80 ℃ for standby. The wet bacterial yield is 10.98 g/L.

Example 4: purification of Chemotaxis recombinant protein

(I) breaking the bacteria

Taking 10-100 g of the thallus obtained by fermentation, and mixing the thallus according to the mass (g): buffer A1 was added to the mixture at a volume (mL) ratio of 1:15, and the bacterial cells were sheared and suspended at 4 ℃ using a shear. The buffer solution A1 comprises sodium chloride 1.17%, sodium carbonate 0.03%, sodium bicarbonate 0.18%, pH8.0, and water as solvent.

The high pressure homogenizer (AH-1500, ATS Industrial systems, Inc.) tube was flushed with RO water. And opening the low-temperature refrigeration system for precooling for later use. And adding the pre-cooled suspension bacterium liquid into a high-pressure homogenizer, breaking the bacterium for 7-8 times under the condition of 480-520 bar, and then taking a whole bacterium sample.

And filling the liquid after the bacteria breaking into a centrifugal barrel, centrifuging for 30min at 10000g at the centrifugal temperature of 8 ℃, and collecting supernatant, namely supernatant 1.

Filtering the supernatant 1 by using a vacuum suction filter pump, collecting the filtered supernatant as a supernatant 2, and taking a supernatant sample.

The pellet was suspended in buffer A1, and the sample was taken after suspension and named pellet.

(II) affinity chromatography purification of recombinant protein Ni packing containing tag Chemotaxis

1. Affinity chromatography conditions:

(1) an instrument system: APPS200D purification System (Suzhou Tech Co., Ltd.)

(2) Filling: Ni-NTA

(3) Specification of a purification column: 50mm x 200mm

(4) Column packing volume: 100mL

2. The packing was equilibrated with buffer a1 to conductance, uv-equilibration.

3. And (3) loading the supernatant 2 at the low temperature of 4 ℃, wherein the loading flow rate is 10mL/min, and after loading 1 column volume, taking the flow-through, namely the flow-through 1.

4. First washing:

(1) after the sample loading is finished, taking flow-through, namely flow-through 2.

(2) And washing with a buffer solution A1, wherein the washing volume is 1-2 column volumes, and the flow rate is 15 mL/min.

5. And (3) second washing: after washing with the buffer A1, washing with the buffer A2 until the UV value is less than 100mAu and the flow rate is 15 mL/min. Buffer a2 consisted of: 2.92% of sodium chloride, 0.03% of sodium carbonate, 0.18% of sodium bicarbonate, 0.136% of imidazole, pH8.0 and water as a solvent.

6. And (3) washing for the third time: after washing with the buffer A2, washing with the buffer A3 until the UV value is less than 70mAu and the flow rate is 15 mL/min. Buffer a3 consisted of: 1.17% of sodium chloride, 0.03% of sodium carbonate, 0.18% of sodium bicarbonate, 0.340% of imidazole, pH8.0 and water as a solvent.

7. And (3) elution: after washing with buffer A3, elution was carried out with buffer A4 at a flow rate of 15 mL/min. The sample collection is started when the UV value is greater than 250mAu, and stopped when the UV value is less than 250 mAu. Buffer a4 consisted of: 1.17% of sodium chloride, 0.03% of sodium carbonate, 0.18% of sodium bicarbonate, 1.700% of imidazole, pH8.0 and water as a solvent. The sample is a recombinant protein containing a label Chemotaxis, and the sample is taken for subsequent SDS-PAGE identification and is named as sample 1, and the result is shown in figure 5;

(III) recombinant protein enzyme digestion containing tag Chemotaxis

The recombinant protein containing the tag Chemotaxis was diluted 5-fold with buffer a 1.

And (3) carrying out centrifugal ultrafiltration concentration on the diluted recombinant protein containing the tag Chemotaxis by using a 10KD ultrafiltration tube under the centrifugal condition of 4000g for 15 min.

Taking out the PP enzyme from an ultra-low temperature refrigerator at minus 80 ℃, and unfreezing the PP enzyme at 4 ℃ for later use.

And adding 10-30 mL of PP enzyme into the concentrated recombinant protein containing the tag Chemotaxis, and performing enzyme digestion at 4 ℃ overnight.

Samples after overnight digestion were taken for subsequent SDS-PAGE identification, this being sample 2.

(IV) purifying the Chemotaxis recombinant protein by Ni column filler affinity chromatography

The filler was equilibrated with buffer a1 to conductance, uv-equilibration. The equipment, packing and purification column used in this step are the same as above.

And (3) loading the overnight enzyme-cleaved recombinant protein solution containing the tag Chemotaxis at the low temperature of 4 ℃, wherein the loading flow rate is 10 mL/min.

The sample collection was started when the UV value was greater than 10 mAU.

After the loading is finished, the loading of the buffer A1 is continued, and the loading flow rate is 10 mL/min.

And stopping collecting the sample when the ultraviolet value is less than 10 mAu.

(V) Chemotaxis recombinant protein concentration

And (3) carrying out centrifugal ultrafiltration concentration on the Chemotaxis recombinant protein sample by using a 10KD ultrafiltration tube, wherein the centrifugal condition is 4000g and 15 min.

Concentrating the Chemotaxis recombinant protein to 5-15 mL, and storing in a-80 ℃ ultra-low temperature refrigerator for later use.

And taking the concentrated Chemotaxis recombinant protein sample for subsequent SDS-PAGE identification, wherein the sample is sample 3.

Example 5: chemotaxis recombinant protein SDS-PAGE identification

15% SDS-PAGE was prepared.

The whole strain, the supernatant, the precipitate, the flow through 1, the flow through 2, the sample 1, the sample 2 and the sample 3 are respectively added with 10 mu L of 5 xSDS-PAGE Loading Buffer, and the mixture is subjected to boiling water bath for 5 min.

After the boiling water bath, SDS-PAGE was added in an amount of 10. mu.L per well, and the amount of Protein Marker added was 3. mu.L.

After sample adding, firstly adjusting the voltage to 80V, and carrying out electrophoresis for 15 min; then the voltage is adjusted to 220v, and electrophoresis is carried out for about 40 min.

And after the electrophoresis is finished, dyeing and decoloring by adopting a Coomassie brilliant blue method.

The results were observed under an imaging system after the decolorization was completed, and the results are shown in FIG. 6.

Example 6: content determination of Chemotaxis recombinant protein

And (3) carrying out protein content determination by adopting a forinophenol method.

And estimating the concentration of the detected sample according to a Chemotaxis recombinant protein SDS-PAGE electrophoretogram.

The measured concentration of the Chemotaxis recombinant protein is 3.70mg/mL, and the correlation coefficient R of a standard curve²When the yield is 0.9940, the recovery rate is 95.5%.

Example 7: preparation of genetically engineered vaccine of helicobacter pylori Chemotaxis

(I), adjuvant LT (B)₅Gene construction

1、LT(B)₅Cloning of genes

LT(B)₅The protein amino acid sequence is selected from SEQ ID NO.16, and the nucleotide sequence is shown as SEQ ID NO. 17.

LT(B)₅The gene was synthesized by Shanghai Czeri bioengineering, Inc.

Designing LT (B) according to the principle of primer design₅Corresponding primers of the gene are added with restriction enzyme cutting sites NdeI and XhoI. The primer sequences are as follows:

2、LT(B)₅the gene was ligated with pET28a plasmid

A mixture of LT (B)₅The gene and pET28a were subjected to double digestion at NdeI and XhoI sites, and the digestion system and the procedures were as described in the specification. NedI/NcoI was from Baori physicians technology (Beijing) Inc.

And (3) performing 1.5% agarose gel electrophoresis on the products subjected to double enzyme digestion respectively, and observing the result under an imaging system after the electrophoresis is finished.

Cutting the gel to recover the required fragment, and performing agarose gel recovery operation.

Double cleaved LT (B)₅The gene was ligated to pET28a plasmid, and the ligation system and procedure were as described in the specification. Ligase (Ligation high) was from Shanghai Biotech, Inc., Toyobo.

3、pET28a-LT(B)₅Transformation of DH5a

The ligated product was transformed into E.coli DH5a competent cells, and the transformation system and procedure were as described in the specification. Coli DH5a competent cells were obtained from Baori physician technology (Beijing) Ltd.

The transformation product was added to 500. mu.L of LB liquid and cultured with shaking at 220rpm at 37 ℃ for 1h, which was culture 7. The composition of LB medium was the same as above.

100 μ L of culture 7 was spread on LB agar plates and cultured overnight at 37 ℃. LB agar plates were composed as above.

Colonies were picked on overnight-cultured LB agar plates for general PCR identification with primers F-B01, R-B01, TaKaRa Ex Taq source, general PCR identification system and procedures as described in TaKaRa instructions.

The colony PCR-identified positive bacteria were inoculated in LB-1 medium and cultured overnight at 37 ℃ with shaking at 220rpm, this being culture 8. The composition of LB-1 medium is the same as above.

And (3) performing seed preservation on the culture 8, wherein the seed preservation parameters are that the culture 8: the ratio of 30% glycerol was 1:1, and the storage conditions were-80 ℃.

The overnight culture 8 was extracted with a plasmid designated pET28a-LT (B)₅. The recombinant plasmid DNA extraction kit and the plasmid extraction program are shown in the specification.

The extracted plasmid is identified by common PCR, the primers are F-B01 and R-B01, and the identification system and the program are shown in the specification.

The plasmid identified by common PCR is stored at-20 ℃ for later use.

4、pET28a-LT(B)₅Conversion BL21(DE)₃

Mixing pET28a-LT (B)₅Plasmid transfer into Escherichia coli BL21(DE)₃Competent cell, largeEnterobacter BL21(DE)₃Competent cells were obtained from Baori physician technology (Beijing) Ltd, and transformation systems and procedures were described in the specification.

The transformation product was added to 500. mu.L of LB liquid and cultured with shaking at 220rpm at 37 ℃ for 1 hour, which was culture 9. The composition of LB medium was the same as above.

50 μ L of culture 9 was spread on LB agar plates and incubated overnight at 37 ℃. LB agar plates were composed as above.

Colonies were picked on overnight-cultured LB agar plates for general PCR identification, TaKaRa Ex Taq source, general PCR identification system and procedure were as above.

Mixing 10 μ L of colony PCR amplification solution with 10 × loadingbuffer 1 μ L, performing 1.5% agarose gel electrophoresis with electrophoresis parameters of 220V for 30min, and observing the result under an imaging system after the electrophoresis is finished, wherein the result is shown in FIG. 7.

The colony PCR-identified positive bacteria were inoculated in LB-1 medium and cultured overnight at 37 ℃ with shaking at 220rpm, this being culture 10. The composition of LB-1 medium is the same as above.

Culture 10 was seed preserved with overnight culture: the ratio of 30% glycerol is 1:1, and the storage condition is-80 deg.C

(II), adjuvant LT (B)₅Escherichia coli BL21(DE)₃Fermentation of

1、pET28a-LT(B)₅Inoculating and culturing Escherichia coli

The constructed pET28a-LT (B)₅Taking out the engineering bacteria of the escherichia coli from an ultralow temperature refrigerator at minus 80 ℃, inoculating the engineering bacteria into an LB-1 culture medium, and culturing overnight at constant temperature of 37 ℃ and 220 rpm. The composition of LB-1 medium is the same as above.

2、pET28a-LT(B)₅Escherichia coli 10L fermentation tank culture

The engineering bacteria cultured overnight are taken out and inoculated in a 10L fermentation tank according to the inoculation proportion of 10 percent, the fermentation medium is TB medium, and the inoculation volume is 5L. Culturing at 37 deg.C and dissolved oxygen concentration above 30% for 2 hr. The TB medium consists of: 0.2312% of monopotassium phosphate, 1.2540% of dipotassium phosphate, 2.4000% of yeast extract, 1.2000% of tryptone, 0.4000% of glycerol, 0.0500% of antifoaming agent and water as solvent.

3、pET28a-LT(B)₅Escherichia coli transferred 100L fermentation tank expanded culture

The engineering bacteria cultured by transfer culture are inoculated into a 100L fermentation tank according to the inoculation proportion of 10 percent, the fermentation medium is TB medium, and the inoculation volume is 55L. Culturing at 37 deg.C and dissolved oxygen concentration above 30% for 6 hr. TB medium was composed as above.

4、pET28a-LT(B)₅Induced expression of Escherichia coli

When the amplification culture is finished, setting the induction temperature to be 16 ℃, and adding 0.8mM IPTG for induction expression, wherein the induction time is 10 h.

5、pET28a-LT(B)₅Collecting Escherichia coli

And (3) when induction is finished, centrifugally collecting thalli by using a centrifugal machine, wherein centrifugal parameters are as follows: 10000g, 8 ℃, 10min, and after the centrifugation, the thalli are stored at minus 80 ℃ for standby. The yield of the bacteria is 120g/L wet bacteria.

(III), adjuvant LT (B)₅Recombinant protein purification

1. Breaking bacteria

Taking 300g of the thallus obtained by fermentation, and mixing the thallus according to the mass (g): buffer B1 was added to the mixture at a volume (mL) ratio of 1:15, and the bacterial cells were sheared and suspended at 4 ℃ using a shear. The buffer solution B1 comprises disodium ethylene diamine tetraacetate 0.115%, sodium chloride 1.17%, sodium carbonate 0.03%, sodium carbonate 0.18%, and glycerol 1.25%.

And (3) filling the liquid after the bacteria breaking into a centrifugal barrel, centrifuging for 30min at 10,000 g, wherein the centrifugation temperature is 8 ℃, and collecting supernatant, namely supernatant 1.

The pellet was suspended in buffer B1, and the sample was taken after suspension and named pellet.

2、LT(B)₅Filler affinity chromatography purification of recombinant protein D-Galactose

(1) Affinity chromatography conditions: the instrument system is an APPS200D purification system (Rivier technologies, Suzhou) and the filler is D-Galactose filler, the specification of the purification column is 50mm multiplied by 200mm, and the packed volume of the column is 100 mL.

(2) The packing was equilibrated with buffer B1 to conductance, uv-equilibration.

(3) And (3) loading the supernatant 2 at the low temperature of 4 ℃, wherein the loading flow rate is 30mL/min, and after loading 1 column volume, taking the flow-through, namely the flow-through 1.

(4) After the sample loading is finished, taking flow-through, namely flow-through 2.

(5) Washing with buffer B1 at flow rate of 50mL/min until the UV value is less than 30 mAu.

(6) Elution was performed with buffer B2 at a flow rate of 50 mL/min. The sample collection is started when the UV value is greater than 100mAu, and stopped when the UV value is less than 100 mAu. Buffer B2 consisted of: disodium ethylenediamine tetraacetate 0.115%, sodium chloride 1.17%, sodium carbonate 0.03%, sodium carbonate 0.18%, glycerin 1.25% and D-galactose 4.50%. pH8.0, and the solvent is water.

The elution sample was LT (B)₅The recombinant protein was sampled and identified by subsequent SDS-PAGE, designated sample 1.

3、LT(B)₅The recombinant protein D-Galactose was purified by pad affinity chromatography as shown in FIG. 8.

(IV), adjuvant LT (B)₅Recombinant protein SDS-PAGE identification

15% SDS-PAGE was prepared.

The whole bacteria, supernatant, precipitate, flow through 1, flow through 2, and sample 1 were each 40. mu.L, and 10. mu.L of 5 xSDS-PAGE Loading Buffer was added to each sample, and the mixture was subjected to boiling water bath for 15 min.

The results were observed under an imaging system after the decolorization was completed, and the results are shown in FIG. 9.

(V), adjuvant LT (B)₅Recombinant protein content determination

According to LT (B)₅The SDS-PAGE electrophoretogram of the recombinant protein is estimated to detect the concentration of the sample.

LT(B)₅The measured concentration of the recombinant protein is 2.3mg/mL, and the correlation coefficient R of a standard curve²The recovery rate is 100.8% when the yield is 0.9945.

(VI), Chemotaxis recombinant protein and LT (B)₅Preparation of vaccine by physical mixing of recombinant proteins

Mixing Chemotaxis recombinant protein, LT (B)₅The recombinant protein was thawed by standing at 4 ℃.

The thawed Chemotaxis recombinant protein, LT (B)₅The recombinant proteins were mixed at a dose of 1 mg/mouse.

The volume of mice immunized was 1 mL/mouse.

Example 12: oral immune animal of helicobacter pylori Chemotaxis genetic engineering vaccine

(I) oral gavage immune animals

1. Experimental animals: 6-week-old female Balb/c mice, 90, 18g + -2 g. Mice were grouped by random grouping, 10 mice/cage, since purchase. 30 immunization groups, 30 infection groups and 30 blank control groups. The immunization experiment was carried out by feeding for 1 day.

2. Chemotaxis vaccine composition: chemotaxis recombinant protein antigen 1mg/mL, LT (B)₅The recombinant protein is 1mg/mL, and the solvent is: 1.17% of sodium chloride, 0.03% of sodium carbonate, 0.18% of sodium bicarbonate and 5mL/100mL of glycerol, wherein the pH value of the solution is 8.0.

3. Infection group (adjuvant control group) immunization composition: LT (B)₅1mg/mL, solvent: 1.17% of sodium chloride, 0.03% of sodium carbonate, 0.18% of sodium bicarbonate and 5mL/100mL of glycerol, wherein the pH value of the solution is 8.0.

4. Oral gavage immunization procedure: the total immunization times are 3 times, and the immunization time points are 0 day, 7 days and 28 days.

5. Before immunization, the patients need to be fed and water-free 24 hours in advance.

6. Oral gavage and immunization:

(1) before immunization, the helicobacter pylori vaccine is taken out from the temperature of minus 80 ℃, placed in a refrigerator at the temperature of 4 ℃ and unfrozen for standby.

(2) Aspirating the Chemotaxis vaccine with a sterile syringe at an immunizing dose of 1mg Chemotaxis antigen per individual LT (B)₅Is 1 mg/piece.

(3) The mice are infused with 1mL of the vaccine by intragastric administration for 3 times for each immunization, and the time interval between each oral administration and intragastric administration does not exceed 45 min.

(4) After the immunization is finished, the water is recovered for 2 h.

(5) Each immunization protocol was identical.

(II) oral gavage infection of helicobacter pylori after last immunization

Performing challenge experiment with oral administration of helicobacter pylori SS1 viable bacteria 10 days after last immunization, wherein the infection dose of each mouse is 4 × 10⁶CFU。

(III) saliva and blood sample collection after the end of the last immunization

1. Mouse saliva sample Collection

Saliva was collected from the mice at day 10 and day 38 after the end of the last immunization.

Before saliva collection, mice need to be deprived of food and water for 24 hours.

Before collecting mouse saliva, injecting pilocarpine 20uL into mouse abdominal cavity at 5mg/mL, collecting saliva, and storing at-80 deg.C for use.

2. Mouse blood sample Collection

Tail vein blood was collected from the mice on day 10 after the end of the last immunization, and orbital vein blood was collected from the mice on day 38.

3. Before saliva collection, mice need to be deprived of food and water for 24 hours.

4. Standing the collected blood at room temperature for 4h, centrifuging for 2min at 3000g, sucking supernatant, repeating the above operation once again, and storing the separated serum at-80 deg.C for use.

(IV) detection of saliva IgA and serum IgG sample by Elisa indirect method

1. Elisa assay preparation

The confining liquid consists of: 0.01M PBS, 1.5% BSA, solvent water.

The PBST wash consisted of: 0.01M PBS, 0.05mL/100mL TWEEN-20, and water as solvent.

The antibody dilutions were composed as follows: 0.01M PBS, 0.05mL/100mL TWEEN-20, 0.5% BSA, solvent water.

The substrate buffer composition was as follows: 1.4 percent of disodium hydrogen phosphate, 1.5 percent of citric acid monohydrate and water as a solvent.

The 2M sulfuric acid composition was as follows: concentrated sulfuric acid of 11.22mL/100mL, and water as solvent.

1mg/ml TMB composition was as follows: TMB 0.1.5%, the solvent was DMSO.

The color developing solution comprises the following components: 1mg/ml TMB: substrate buffer: the preparation volume ratio of 30 percent hydrogen peroxide is 100:900: 1.

Chemotaxis antigen coated elisa plate: the ELISA plate was coated with 2ug/mL of the immune antigen, incubated at 37 ℃ for 2h, and washed three times with PBST wash. Add the blocking solution to the above ELISA plate at 300. mu.L/well, put in a refrigerator at 4 ℃ and block overnight. Washing the ELISA plate with PBST washing liquid for three times, naming the ELISA plate as ELISA plate 1 and storing in 4 deg.C refrigerator for standby.

2. Serum IgG sample Elisa detection

Diluting the serum sample with an antibody diluent at a ratio of 1:800, adding 100 mu L/hole into an ELISA plate 1, incubating for 45min at 37 ℃, washing the plate with a PBST washing solution for three times, and naming the ELISA plate as an ELISA plate 2.

Diluting goat anti-mouse IgG secondary antibody with antibody diluent 1:10000, adding 100 mu L/hole into an ELISA plate 2, incubating at 37 ℃ for 45min, washing the plate with PBST washing solution for three times, and naming the ELISA plate as an ELISA plate 3.

Adding developing solution into enzyme-linked immunosorbent assay plate 3 at a concentration of 100 μ L/well, incubating at 37 deg.C for 15min, and adding 2M H at a concentration of 50 μ L/well₂SO₄The enzyme label plate is named as enzyme label plate 4.

And (3) placing the ELISA plate 4 into an ELISA analyzer, selecting OD450 for detection, storing detection data and carrying out subsequent analysis.

3. Saliva IgA sample Elisa detection

Diluting the saliva sample with an antibody diluent at a ratio of 1:5, adding 100 mu L/well into an ELISA plate 1, incubating at 37 ℃ for 45min, washing the plate with a PBST washing solution for three times, and naming the ELISA plate as an ELISA plate 5.

Diluting goat-anti-mouse IgA secondary antibody with an antibody diluent 1:5000, adding an ELISA plate 5 into a hole with the concentration of 100 mu L, incubating at 37 ℃ for 45min, washing the plate with PBST washing solution for three times, and naming the ELISA plate as an ELISA plate 6.

Adding developing solution into enzyme-linked immunosorbent assay plate 6 at a concentration of 100 μ L/well, incubating at 37 deg.C for 15min, and adding 2M H at a concentration of 50 μ L/well₂SO₄The enzyme label plate is named enzyme label plate 7.

And (3) placing the ELISA plate 7 into an ELISA analyzer, selecting OD450 for detection, storing detection data and carrying out subsequent analysis.

4. Results of measurement of salivary IgA and serum IgG titer

And (3) judging a detection result: positive was defined as a sample (immune group)/negative (infectious group) value ≧ 2.1. Positive mice for the detection of titer in the infected group/mice in the infected group x 100% were defined as the positive conversion rate.

(1) The result of the saliva IgA titer detection: the positive conversion rate of salivary IgA (1:4) titer detection at the end point of the judgment of the protection rate of the mouse immune group was 40%, as shown in fig. 10.

(2) Serum IgG titer test results: the positive conversion rate of the serum IgG (1:800) titer detection at the end point of the determination of the protection rate of the mouse immune group was 70%, as shown in FIG. 10.

(3) The detection results of the saliva IgA and serum IgG titer show that the Chemotaxis vaccine has good immunogenicity, can induce an organism to generate immune response, and provides support for the Chemotaxis vaccine for removing the helicobacter pylori colonization in the stomach.

(V) Chemotaxis vaccine protection results

1. Detecting the helicobacter pylori infection colonization rate of each group of mice by a plate culture method on the 38 th day after the end of the last immunization, and calculating the Chemotaxis vaccine protection rate. The protection rate is (number of immunization groups × infection rate of infection group — number of infection groups)/(number of immunization groups × infection rate of infection group) × 100/100.

2. The culture of helicobacter pylori is carried out by referring to the conventional conditions, formula and operation method in the field.

3. Helicobacter pylori plate culture judgment standard:

(1) and colony morphology identification standard: judging whether the helicobacter pylori is positive or not by the characteristic bacterial colony which grows on the flat plate and has the diameter of 0.1-0.5 mm and the size of a transparent needle point, and indicating the bacterial colony by a plus sign; when no colonies of the above morphology were grown or not on the plate, it was judged as helicobacter pylori negative and indicated by "-"; when bacteria of a suspected H.pylori colony morphology grew on the plate, they are indicated by ". dot..

(2) And the standard of quick urease experimental identification: dripping a proper amount of urease solution on the flat bacterial colony, judging that the bacterial colony is positive by urease if the bacterial colony becomes red, and judging that the bacterial colony is positive by helicobacter pylori and indicated by a plus sign; if the urea catalase reaction of the bacterial colony does not change into red or does not change into color, judging the bacterial colony to be helicobacter pylori negative and indicating by a mark; when the color of the urea catalase reaction of the colony is not obvious, the colony is judged to be suspected helicobacter pylori and is indicated by a mark.

(3) Fast gram staining microscopy identification standard: and (3) performing microscopic examination when the colony morphology and the rapid urease experiment are suspected, wherein the microscopic examination standard is as follows: when the bacteria are purple, the shape is spiral bending, and the tail end is blunt, the bacterial colony is judged to contain the helicobacter pylori, and the mark is indicated by a plus sign; on the contrary, if the microscopic bacteria are not purple, and the shape has no spiral bending and blunt ends, the helicobacter pylori is judged to be negative and is indicated by a mark of "-"; if this test is not performed, it is indicated by a "/" sign.

4. Helicobacter pylori plate culture judgment standard: when one or more than one colony grows on the flat plate, carrying out colony morphology identification, a rapid urease experiment and a rapid gram staining microscopy, and when the colony morphology identification and the rapid urease experiment are positive or the rapid gram staining microscopy is positive, judging that the colony is helicobacter pylori, and simultaneously judging that the helicobacter pylori is infected and planted in the stomach of the mouse successfully; when the colony morphology identification and the rapid urease experiment are both negative or negative in rapid gram staining microscopy, the colony is judged not to be helicobacter pylori, and meanwhile, the helicobacter pylori infection colonization in the stomach of the mouse is judged to be unsuccessful.

5. The planting rate detection results of continuous 3 rounds (10/round) of animal protection experiments of the Chemotaxis vaccine immunization group are shown in the table 3-5:

TABLE 3

TABLE 4

TABLE 5

6. As the morphological identification and the rapid urease test of helicobacter pylori colonies can be directly carried out from the culture plate, the rapid gram staining microscopy is not carried out.

7. According to the results of plate culture, the average colonization rate of each group in 3 successive animal protection experiments is shown in table 6:

TABLE 6

Group of	Rate of permanent planting
		Immunization group	83.3％(23/30)
Infection with viral infectionGroup of	96.7％(29/30)
		Blank control group	0％(0/30)

According to the planting rate results, the protection rate is shown in table 7:

TABLE 7

Group of	Rate of protection
		Immunization group	20.7％(6/29)
Infectious group	0％(0/29)
		Blank control group	0％(0/30)

As can be seen from the above table, the average protection rate of the immunization group in 3 rounds was 20.7%, and the average protection rate of the infection group in 3 rounds was 0%.

Therefore, the Chemotaxis recombinant protein has good immunogenicity, can induce a mouse to generate mucosal immune response, can eliminate the colonization of helicobacter pylori SS1 strains in the stomach of the mouse, and can be prepared into a helicobacter pylori subunit vaccine by a mucosal adjuvant and the Chemotaxis recombinant protein to prevent helicobacter pylori infection.

Through the embodiment, the fermentation and purification steps of the Chemotaxis recombinant protein are simple, the obtained Chemotaxis recombinant protein is high in yield and strong in immunogenicity, and the Chemotaxis recombinant protein can be further applied to prevention of helicobacter pylori infection.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> WUDOUYIMIAO Biotechnology Ltd

Application of <120> helicobacter pylori chemotactic factor chemotaxis gene

<160> 19

<170> SIPOSequenceListing 1.0

<210> 1

<211> 933

<212> DNA

<213> Helicobacter pylori (Helicobacter pylori)

<400> 1

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caagagatta tccccatgcc cacccttttt gaatacccca cgaatttgga ttacattatc 180

ggcgtgtttg atttgcgctc cacgatcatt ccgcttatag acttggctaa atggataggg 240

attgtcccag ataaaagcaa ggaaaacgaa aaaatcgtca ttatcacgga atttaataat 300

gtcaaattgg gctttttagt ccattccgct aggcgtatca ggcgcattag ctggaaagat 360

gtggagcctg catcctttag tgcctctaat agcatcaata aagaaaatat caccggcacg 420

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<212> PRT

<213> Helicobacter pylori (Helicobacter pylori)

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Ala Glu Lys Thr Ala Asn Asp Leu Lys Leu Ser Glu Ile Glu Leu Val

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Asp Phe Arg Ile Tyr Gly Met Gln Glu Gly Val Pro Tyr Glu Gly Ile

20 25 30

Tyr Gly Ile Asn Val Ala Lys Val Gln Glu Ile Ile Pro Met Pro Thr

35 40 45

Leu Phe Glu Tyr Pro Thr Asn Leu Asp Tyr Ile Ile Gly Val Phe Asp

50 55 60

Leu Arg Ser Thr Ile Ile Pro Leu Ile Asp Leu Ala Lys Trp Ile Gly

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Ile Val Pro Asp Lys Ser Lys Glu Asn Glu Lys Ile Val Ile Ile Thr

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Glu Phe Asn Asn Val Lys Leu Gly Phe Leu Val His Ser Ala Arg Arg

100 105 110

Ile Arg Arg Ile Ser Trp Lys Asp Val Glu Pro Ala Ser Phe Ser Ala

115 120 125

Ser Asn Ser Ile Asn Lys Glu Asn Ile Thr Gly Thr Thr Arg Ile Glu

130 135 140

Asn Asp Lys Thr Leu Leu Ile Leu Asp Leu Glu Ser Ile Leu Asp Asp

145 150 155 160

Leu Lys Leu Asn Glu Asp Ala Lys Asn Thr Lys Asp Thr Pro Lys Glu

165 170 175

Arg Phe Glu Gly Glu Val Leu Phe Leu Asp Asp Ser Arg Thr Ala Arg

180 185 190

Lys Thr Leu Lys Asn His Leu Ser Lys Leu Gly Phe Ser Ile Thr Glu

195 200 205

Ala Val Asp Gly Glu Asp Gly Leu Asn Lys Leu Glu Met Leu Phe Lys

210 215 220

Lys Tyr Gly Asp Asp Leu Arg Lys His Leu Lys Phe Ile Ile Ser Asp

225 230 235 240

Val Glu Met Pro Lys Met Asp Gly Tyr His Phe Leu Phe Lys Leu Gln

245 250 255

Lys Asp Pro Arg Phe Ala Tyr Ile Pro Val Ile Phe Asn Ser Ser Ile

260 265 270

Cys Asp Asn Tyr Ser Ala Glu Arg Ala Lys Glu Met Gly Ala Val Ala

275 280 285

Tyr Leu Val Lys Phe Asp Ala Glu Lys Phe Thr Glu Glu Ile Ser Lys

290 295 300

Ile Leu Asp Lys Asn Ala

305 310

<210> 3

<211> 7

<212> PRT

<213> Helicobacter pylori (Helicobacter pylori)

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His His His His His His His

1 5

<210> 4

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys Gly

1 5 10 15

Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr Gly

20 25 30

Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe Pro

35 40 45

Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala His

50 55 60

Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr

65 70 75 80

Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala

85 90 95

Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala

100 105 110

Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr

115 120 125

Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys

130 135 140

Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu

145 150 155 160

Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr

165 170 175

Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly Leu

180 185 190

Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp Thr

195 200 205

Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala Met

210 215 220

Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys Val

225 230 235 240

Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys

245 250 255

Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn

260 265 270

Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu

275 280 285

Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala Leu

290 295 300

Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr

305 310 315 320

Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln Met

325 330 335

Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala Ser

340 345 350

Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr

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<210> 6

<211> 1098

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

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<213> Artificial Sequence (Artificial Sequence)

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Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr

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Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr

20 25 30

Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly

35 40 45

Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu

50 55 60

Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met

65 70 75 80

Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly

85 90 95

Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys

100 105 110

Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met

115 120 125

Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly

130 135 140

Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val

145 150 155 160

Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val

165 170 175

Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu

180 185 190

Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr

195 200 205

Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Glu Val Leu Phe

210 215 220

Gln Gly Pro Leu

225

<210> 8

<211> 545

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcccctatac taggttattg gaaaattaag ggccttgtgc aacccactcg acttcttttg 60

gaatatcttg aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg tgataaatgg 120

cgaaacaaaa agtttgaatt gggtttggag tttcccaatc ttccttatta tattgatggt 180

gatgttaaat taacacagtc tatggccatc atacgttata tagctgacaa gcacaacatg 240

ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc ggttttggat 300

attagatacg gtgtttcgag aattgcatat agtaaagact ttgaaactct caaagttgat 360

tttcttagca agctacctga aatgctgaaa atgttcgaag atcgtttatg tcataaaaca 420

tatttaaatg gtgatcatgt aacccatcct gacttcatgt tgtatgacgc tcttgatgtt 480

gttttataca tggacccaat gtgcctggat gcgttcccaa aattagtttg ttttaaaaaa 540

cgtat 545

<210> 9

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

catcatcatc atcatcataa aatcgaagaa ggtaaa 36

<210> 10

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ataacctagt ataggggaag tctgcgcgtc tttcag 36

<210> 11

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ctgaaagacg cgcagacttc ccctatacta ggttat 36

<210> 12

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

catgccatgg acaggggccc ctggaacag 29

<210> 13

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ggaattccat atgcatcatc atcatcatca tcat 34

<210> 14

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

catgccatgg cagaaaaaac agctaacg 28

<210> 15

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ggaattctta tgcattcttg tctaaaatct tag 33

<210> 16

<211> 124

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Met Asn Lys Val Lys Cys Tyr Val Leu Phe Thr Ala Leu Leu Ser Ser

1 5 10 15

Leu Cys Ala Tyr Gly Ala Pro Gln Ser Ile Thr Glu Leu Cys Ser Glu

20 25 30

Tyr Arg Asn Thr Gln Ile Tyr Thr Ile Asn Asp Lys Ile Leu Ser Tyr

35 40 45

Thr Glu Ser Met Ala Gly Lys Arg Glu Met Val Ile Ile Thr Phe Lys

50 55 60

Ser Gly Ala Thr Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp

65 70 75 80

Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Thr

85 90 95

Tyr Leu Thr Glu Thr Lys Ile Asp Lys Leu Cys Val Trp Asn Asn Lys

100 105 110

Thr Pro Asn Ser Ile Ala Ala Ile Ser Met Glu Asn

115 120

<210> 17

<211> 375

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atgaacaaag tcaaatgtta tgttttattt acggcgttac tgtcctctct gtgtgcatac 60

ggagctccgc agtctattac agaactgtgt tcggaatatc gcaacacaca aatttatacg 120

attaatgaca agattctgtc atatacggaa tcgatggcag gcaaacgcga aatggttatc 180

attacattta agagcggcgc aacatttcag gtcgaagtcc cgggcagtca acatattgac 240

tcccaaaaaa aagccattga acgcatgaag gacacattac gcatcacata tctgaccgag 300

accaaaattg ataaattatg tgtatggaat aataaaaccc cgaattcaat tgcggcaatc 360

agtatggaaa actag 375

<210> 18

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ggaattccat atgaacaaag tcaaatgtta tg 32

<210> 19

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ccgctcgagc tagttttcca tactgattg 29

Claims

1. An application of a helicobacter pylori chemotactic factor chemotaxis gene in preparing a helicobacter pylori vaccine is characterized in that the nucleotide sequence of the chemotaxis gene is shown as SEQ ID NO. 1.

2. An application of helicobacter pylori chemotactic factor chemotaxis recombinant protein in preparing helicobacter pylori vaccine, wherein the amino acid sequence of the chemotaxis recombinant protein is shown as SEQ ID NO. 2.

3. An expression vector for expressing helicobacter pylori chemotactic factor chemotaxis recombinant protein, which is characterized by comprising: chemotaxis protein, His-tag, pET29b plasmid, GST protein and MBP protein.

4. A construction method of an expression vector for expressing helicobacter pylori chemotactic factor chemotaxis recombinant protein is characterized by comprising the following steps:

5. A fermentation method for expressing helicobacter pylori chemotactic factor chemotaxis recombinant protein is characterized by comprising the following steps:

6. The fermentation method of claim 5, wherein the TB culture medium comprises: 0.2312% of monopotassium phosphate, 1.2540% of dipotassium phosphate, 2.4000% of yeast extract, 1.2000% of tryptone, 0.4000% of glycerol, 0.0500% of an antifoaming agent and the balance of water.

7. The fermentation method of claim 5, wherein the centrifugation parameters in step 4) are as follows: 10000g, 8 ℃, 10 min.